Differential response to calcium-labelled (44Ca) uptake and allocation in two peach rootstocks in relation to transpiration under in vitro conditions

(1)

Scientia Horticulturae 326 (2024) 112718

Available online 28 November 2023

Differential response to calcium-labelled ( ⁴⁴ Ca) uptake and allocation in two peach rootstocks in relation to transpiration under in vitro conditions

Francisca Carrasco-Cuello

^a^,^b^,^*

, Laia Jen ´ e

^a

, Ramon Dolcet-Sanjuan

^a

, Ana Qui ˜ nones

^c

, Josep Rufat

^b

, Estanis Torres

^a

aIRTA Fruitcentre, Fruticulture Program, Parc AgroBiotech, Lleida, Spain

bIRTA Fruitcentre, Efficient Use of Water in Agriculture Program, Parc AgroBiotech, Lleida, Spain

cIVIA, Centro para el Desarrollo de la Agricultura Sostenible, Moncada, Valencia, Spain

A R T I C L E I N F O Keywords:

Isotope Macronutrients Prunus persica Garnem Rootpac Plant nutrition

A B S T R A C T

Calcium-labelled (⁴⁴Ca) uptake and transport under in vitro GreenTray® temporary immersion bioreactor conditions have been studied related to aeration conditions. For this aim, Rootpac®-20 (RP-20) and Garnem® (G × N) were selected as two main rootstocks used in peach production. Two transpiration conditions, aerated and unaerated, were established for each plant material. ⁴⁴Ca location, plant development and foliar stomata surface were measured after the in vitro culture period. The results showed that aeration improved Ca transport within the shoot, but it did not enhance Ca uptake by the roots. Regarding plant material, G ×N presented a better Ca uptake capacity and concentration. The findings suggest that Ca uptake in the roots is a precise process that is influenced by transpiration. However, it was observed that transpiration and thus the water flux is not the only force promoting Ca uptake by roots. Furthermore, the transport of Ca to the shoot was primarily determined by transpiration, indicating that water flux plays a crucial role in the aboveground movement of Ca. The study also revealed distinct behaviors in Ca uptake and allocation between the different peach rootstocks, emphasizing the importance of considering these factors in the selection process of rootstocks. These findings contribute to our understanding of the mechanisms involved in Ca uptake and transport in peach rootstocks under in vitro conditions. They provide valuable insights for rootstock selection processes and highlight the need for further research in this area.

1. Introduction

Calcium (Ca) has traditionally been used as an example of a nutrient with low phloem mobility in plants (White and Ding, 2023). Due to this characteristic, the upward delivery of Ca occurs withing the transpiration stream through the xylem (Hanger, 1979; Montanaro et al., 2010).

Therefore, the movement and delivery of Ca is strongly influenced by the plant transpiration rate, which also depends on various environmental and orchard conditions such as soil water availability, vapor pressure deficit (VPD) or light interception (Girona et al., 2002; Ayars et al., 2003). When the transpiration is low, the root pressure strongly influences the xylem flow, and consequently is also affected by soil os- motic potential. Therefore, roots are not only involved in Ca uptake, but also in Ca transport, especially when the transpiration rate is low (Schenk et al., 2021).

A recent study reported that increasing the xylem vessel area is a new

putative target in breeding approaches related to enhancing stomatal conductance and improving adaptability to Ca deficiencies in peaches (Aras et al., 2021). By contrast, peach rootstock selection has been traditionally based on plant vigor, fruit quality and soil adaptability (Reig et al., 2016; Iglesias et al., 2019). Additionally, the uptake capacity of mineral nutrients is determined by the rootstock choice, and therefore, its responses under nutritional deficit conditions should be un- derstood and considered in the rootstock selection process (Melgar et al., 2023). In this study, a novel approach to trace Ca uptake and movement in peach rootstocks is described.

Traditionally, plant nutrition studies have employed isotopes to trace nutrients, and therefore, find out easily the uptake, distribution, and partitioning rates. A common technique to investigate Ca uptake is the use of the radioactive isotope ⁴⁵Ca that permitted to trace Ca by its radioactivity (Bonomelli et al., 2022). Even though this technique has been widely used in agriculture, there are some difficulties related to

* Corresponding author.

E-mail address: [email protected] (F. Carrasco-Cuello).

Contents lists available at ScienceDirect

Scientia Horticulturae

journal homepage: www.elsevier.com/locate/scihorti

https://doi.org/10.1016/j.scienta.2023.112718

Received 1 August 2023; Received in revised form 10 November 2023; Accepted 22 November 2023

(2)

Scientia Horticulturae 326 (2024) 112718

human health or environmental risks that promote the development of new approaches. The stable isotope ⁴⁴Ca, although has not been commonly employed in agronomical studies, can provide an easy and safe way to trace Ca, as shown before in apple (Kalcsits et al., 2019). The natural concentration of ⁴⁴Ca is sufficiently low (2.086 %), enabling the identification of the majority of ⁴⁴Ca in plant tissues originating from the applied nutrient solution enriched with a higher concentration of ⁴⁴Ca.

This approach opens new possibilities for gathering insights into calcium nutrition in crops, offering a valuable alternative to radioactive isotopes.

Although the ⁴⁴Ca use should be theoretically the most suitable option to trace Ca, its high price currently makes this option unfeasible in field assays. As an alternative, the ⁴⁴Ca trace under in vitro laboratory conditions is proposed. For this aim, the recently patented temporary immersion system (TIS) bioreactor GreenTray® (Dolcet-Sanjuan and Mendoza, 2018) brings the optimal platform to trace ⁴⁴Ca under in vitro controlled conditions, providing a good stage to evaluate Ca dynamics and accumulation using Ca-labelled fertilizers. Herein the effect of aeration on Ca uptake and movement, using the stable Ca isotope ⁴⁴Ca as a tracer, in two different peach rootstocks grown in the bioreactor

GreenTray® have been studied. This implies a novel approach in plant nutrition studies that will effectively streamline the assessment of nutrient absorption.

2. Materials and methods 2.1. In vitro plant material

Two different Prunus commercial rootstocks were used for the study:

Rootpac® 20 (RP-20) and Garnem® (G ×N) (Fig. 1A). RP-20 rootstock is a hybrid between Prunus cerasifera and Prunus besseyi and G × N rootstock constitutes a hybrid between Prunus dulcis and Prunus persica.

The study was conducted using in vitro propagated shoot-tip cultures of both rootstocks. Both rootstocks were propagated by axillar branch- ing in MS medium (Murashige and Skoog, 1962) with 5 µM benzyla- minopurine (BAP), as described by Iglesias et al. (2004). Two-cm-long nodal segments derived from shoot tip cultures in the multiplication phase were used as initiation explants. Shoot rooting was induced in

½-MS supplemented with 10 µM indole-3-acetic acid (IAA), for 7 days Fig. 1.Plant material (A), GreenTray® (B), air relative humidity inside an unaerated or aerated GreenTray® during a 24 h dark light cycle (C) and GreenTray® operating diagram (D).

F. Carrasco-Cuello et al.

(3)

under 24 ^◦C and dark conditions. Afterwards, shoots were transferred to a ½-MS medium without auxin for root elongation during 7 days under 24 ^◦C and a 16-hour-photoperiod with white-light fluorescents (100–120 µmol m⁻²s⁻¹). The two ½-MS mediums had 50 % MS-salt and macronutrient concentration and were supplemented with Fe-EDTA (10 mM) and CuSO4⋅5H2O (0.1 g L⁻¹). All media were pH-adjusted to 5.7 using NaOH (1 M) before adding agar (8 g L⁻¹) and were then autoclaved at 121 ^◦C for 20 min. Glass tubes, 15 mm of diameter, with 15 ml of medium each were utilized.

2.2. Culture conditions and experimental design

GreenTray® temporary immersion bioreactors (Patent number ES2763637B1) (Dolcet-Sanjuan and Mendoza, 2018) were used for the development of rooted shoots as a system to evaluate the root uptake and plant transport of Ca (Fig. 1B). A modified liquid MS-medium (Murashinge and Skoog, 1962), pH 5.7 and autoclaved at 121 ^◦C for 20 min (without adding sugar or hormones) was used in the GreenTray®

bioreactor culture system (Cantabella et al., 2022).

The experiment consisted of two different treatments, combining two bioreactor ventilation conditions to force two plant transpiration situations (aerated and unaerated) on both rootstocks. The bioreactor air phase was renewed in the aeration treatment, while in the unaerated there were not a force air renewal of the aerial part.

Fifteen rooted shoots per bioreactor were used. During the plant material establishment to the GreenTray® culture conditions (3 days), all cultures were kept at 24 ±1 ^◦C under a photoperiod of 16 h of cool- white fluorescent light (100–120 µmolm⁻²s⁻¹), and an immersion fre- quency of 2 min every 3 h. Afterwards, the culture medium was replaced with fresh medium of the same composition but with 38.81 % of ⁴⁴CaCl2

and 61.19 % of CaCl2. ⁴⁴CaCl2 was prepared by dissolving ⁴⁴CaCO3

(enriched 97 %, Cambridge Isotope Laboratories, Inc., USA) in concen- trated HCl based on Kalcsits et al. (2017).

Throughout the following 10 days, the culture conditions were the same as in the establishment phase, but one bioreactor for each rootstock were heavily aerated. To study the effect of ventilation and transpiration on Ca accumulation, the bioreactor air phase was renewed in the aeration treatment, while in the unaerated there were not a force air renewal of the aerial part (Fig. 1D). In the aerated regime, a 60-second- long aeration of the GreenTray® culture flask was scheduled every 15 min. While the unaerated culture regime was saturated till 94–99 % of ambient humidity inside the bioreactor, the aerated conditions maintain humidity between 49 % and 91 % (Fig. 1C).

After 10 days of culture on the enriched medium with ⁴⁴Ca, the bioreactor immersion and aeration were stopped, and several measurements on the plantlets were taken to evaluate the differences in the plant growth and Ca nutrition.

2.3. Morphological measurements

Plants were extracted and total plantlet, root and shoot fresh weight and shoot and root length (cm) were measured shortly after collection in each plant. Vegetative fractions were oven-dried (Memmert, ULP 800), at 65 ^◦C until constant weight and the dry material was ground and homogenised using a mortar and pestle till powder. Dry weight (DW, g) was calculated afterwards. Plantlet, root, and shoot fresh and root/shoot index, and shoot and root length (cm) were measured in each plant. For the root length, the longest root was measured in each plant.

Fig. 2. Differential responses of rootstocks G ×N (black circles and continuous lines) and RP-20 (gray squares and dashed lines), under aerated or unaerated conditions (abscissa axis) for Dry weight for root (A), shoot (B) and the whole plantlet (C) and Fresh weight (FW) for root (D), shoot (E) and the whole plantlet (F). A two-way ANOVA was used to test the main effects of conditions (C) and rootstocks (R). Statistical significance was assumed at p <0.05*.

(4)

2.4. Stomatal density and foliar area

The total foliar stomatal surface (FSS) (mm²) and total foliar area (cm²) were determined. Stomatal parameters were measured from the second and fourth totally developed youngest leaf of three plantlets following the methodology described by Zhu et al. (2018). An optical white-light microscope (Leica DM5000B, Leica Microsystems CMS GmbH, Germany) was used to observe the stomatal characteristics under 40 magnifications (40x) and total stomata were counted from three different leaf slides from each leaf. In this experiment, a colourful digital Leica camera (Leica DFC 420) connected with the microscope was used to take images from the different leaf fields and from five individual stomata. Images were analysed using the Application Suite System LEICA and the stomata length and stomata width were measured (µm) to calculate the individual stomatal area using the ellipse area formula [(π

×sl ×sw)], where sl is the stomata length and sw is the stomata width.

The total FSS was determined following the formula [(stomatal density)

×(individual stomatal area) ×(total foliar area)]. The total foliar area was measured with LeafByte application, following the protocol described by Getman-Pickering et al. (2020).

2.5. Calcium analysis

For each bioreactor, three groups of plantlets were established considering their homogeneity and for each group, roots and shoot were separated. Hence, a total of six samples were collected for every

bioreactor (3 from shoots and 3 from roots). Mineral content and isotope analysis were measured at the analytical mass spectrometry department (CACTI) of the University of Vigo. Samples were oven-dried, and the dry material was ground and homogenized using a mortar and pestle till powder. Mineral content determination was achieved by nitric digestion and subsequent dilution of the digestion with mili-Q water. The total Ca concentration (mg g⁻¹DW) was measured with an atomic emission spectrometer with an inductively coupled plasma source (ICP-AES iCAP 6000, Thermo Scientific). The ⁴⁴Ca concentration was determined in a mass spectrometer with a multiple collector and inductively coupled plasma source (MC-ICP MS, Thermo Finnigan Neptune, University of Vigo) following the described methodology by Heuser et al. (2002).

The ⁴⁴Ca excess (%) of each sample was calculated following the formula:

44Ca excess(%) =%⁴⁴Ca− 2.086%

where 2.086 is the percentage of Ca naturally found in nature.

To calculate the ⁴⁴Ca (mg) the following formula was applied:

44Ca(mg) =⁴⁴Ca excess(%) ×DW(mg) ×Ca( mg⋅kg⁻¹)

×10⁴

The Ca derived from the fertilizer (Cadff), which means the pro- portion of Ca in the plant that comes from the fertilizer (in this case from the in vitro culture medium) was calculated following the formula:

Cadff(%) =

44Ca excess

44Ca excess culture medium × 100

Fig. 3. Root length (cm) (A), Shoot length (cm) (B), Foliar area (cm²) (C) and Foliar Stomata Surface (FSS) (mm²) (D) for unaerated and aerated plantlets, in GxN (black circle and continuous lines) and RP-20 (grey square and dashed lines). A two-way ANOVA was used to test the main effects of conditions (C) and rootstocks (R). Statistical significance was assumed at p <0.05*.

(5)

where ⁴⁴Ca excess (%) enrichment in culture medium was of 36.836 %.

2.6. Statistical analysis

The data were analysed via analysis of variance (ANOVA) by using JMP Pro-Software (version 16.0, SAS Institute Inc., Cary, NC). A two- way ANOVA was used to test the main effects of conditions (C), aerated and unaerated, and rootstocks (R), G ×N and RP-20, in roots, shoots and the whole plantlet. Statistical significance was assumed at p levels <0.05.

3. Results

3.1. Morphological and physiological plantlet changes

The root DW (g) was independent of the conditions and the rootstocks. On the other hand, the shoot and total plantlet DW was determined by the conditions, but not by the rootstock. Concretely, shoot DW was significantly higher under the aeration conditions (0.32 g for aerated and 0.19 g for unaerated) as well as the total plantlet DW (0.45 g for aerated and 0.3 g for unaerated) (Fig. 2A, B and C).

Regarding FW (g), the rootstock and the conditions had differentially influenced the parts of the plant. On one hand, G × N presented significantly higher FW values than RP-20 in the roots (1.48 and 0.76 g respectively) and in the whole plantlet (2.72 g for G ×N and 1.95 g for RP-20). On the other hand, the bioreactor aeration significantly increased the FW in the roots (1.35 g for aerated and 0.76 g for unaerated), the shoots (1.54 g for aerated and 0.89 g for unaerated) and the whole plant (2.88 g for aerated and 1.8 g for unaerated) (Fig. 2D, E and

F). Root length (cm) was significantly affected by the interaction between the rootstock and the conditions. While the aeration in G ×N significantly increased root length, the RP-20 aerated and unaerated values were similar. Moreover, only under unaerated conditions there were statistical differences between rootstocks, where the RP-20 presented the longest roots. Apart from that, RP-20 significantly had longer shoots than G ×N (4.65 and 2.83 cm respectively) and shoot growth was enhanced with aeration for both rootstocks (Fig. 3A and B).

What is more, the differences between rootstocks foliar areas (cm²) were significantly increased by aeration of the GreenTray®, especially with G ×N, which doubled the RP-20 leaf surface under aeration conditions (Fig. 3C). Concretely, RP-20 presented a significantly higher stomata density (n^◦stomata/mm²) (p <0.0001) than G ×N, but G ×N significantly presented a biggest stomata size (µm²) (p <0.0001) (data not shown). Consequently, G ×N presented a higher FSS (mm²) than RP- 20 under aerated conditions, and under unaerated conditions both rootstocks presented a similar FSS (Fig. 3D).

3.2. Ca concentration and content

Overall, RP-20 presented significantly lower Ca concentration than G ×N, especially under unaerated conditions. Concretely in the roots, Ca concentration, expressed as Ca mg g⁻¹DW, of both rootstocks was influenced by the bioreactor aeration conditions. While G ×N roots presented higher values in unaerated than in aerated conditions, the Ca concentrations for roots of RP-20 were higher for aerated conditions than for unaerated. Regarding shoots, G ×N presented a significantly higher Ca (mg g⁻¹DW) concentration than RP-20, without any Fig. 4. Differential responses of rootstocks G ×N (black circles and continuous lines) and RP-20 (grey squares and dashed lines), under aerated or unaerated conditions (abscissa axis), for Ca (mg g⁻¹DW) (A, B and C) and Ca (mg) (D, E and F) on roots (A and D), shoots (B and E) and the whole plantlet (C and F). A two-way ANOVA was used to test the main effects of conditions (C) and rootstocks (R). Statistical significance was assumed at p <0.05*.

(6)

statistical differences between the conditions. However, no differences between aerated and unaerated conditions were found in total Ca concentration (Fig. 4A, B and C).

Concerning Ca (mg) content, as for Ca concentrations, G ×N presented the highest values, but only statistically different in the roots.

Even more, the G ×N roots doubled the Ca content of RP-20 (0.56 and 0.26 mg respectively) independently from the conditions. Besides that, a higher Ca (mg) content, although not significant, was observed on aerated shoots and whole plantlets for both rootstocks when compared to unaerated (Fig. 4D, E and F).

3.3. ⁴⁴Ca-labelled concentration and content

In general terms, the results for ⁴⁴Ca (mg) and ⁴⁴Ca (mg g⁻¹DW) tended to present a similar pattern to those presented before for non- labelled Ca. G × N generally presented a higher ⁴⁴Ca concentration and content than RP-20. Concretely, ⁴⁴Ca (mg g⁻¹DW) concentration indicated that both rootstocks were differently influenced by the bioreactor aeration conditions. Meanwhile the G ×N roots exhibited the highest ⁴⁴Ca (mg g⁻¹DW) under unaerated conditions, the RP-20 roots presented the highest ⁴⁴Ca concentration under aerated conditions, resulting in an opposite roots response to bioreactor conditions depending on the plant material (Fig. 5A, B and C).

The ⁴⁴Ca (mg) in roots was significantly higher in G ×N than in RP- 20 (0.15 and 0.06 mg respectively), although no statistical differences were found in roots regarding the conditions. Interestingly, aeration did not influence ⁴⁴Ca (mg) in the roots, however, it demonstrated a noticeable effect on the shoots. Under aeration conditions, the ⁴⁴Ca (mg) significantly increased in the shoots (0.13 mg for unaerated and 0.27 mg

for aerated and). Shoot accumulation of ⁴⁴Ca was not different between rootstocks. In general terms, the total plantlet ⁴⁴Ca (mg) was not significantly influenced either by the rootstock or the bioreactor conditions. Although the aerated plant tended to present the highest ⁴⁴Ca (mg) contents (Fig. 5D, E and F).

For ⁴⁴Ca excess (%), the significant differences found in roots were only due to the rootstocks, where G ×N presented the highest values (27.38 % for G ×N and 23.08 % for RP-20). By contrast, in shoots there were significant differences for rootstocks and bioreactor conditions. In this part of the plantlet, RP-20 significantly presented more ⁴⁴Ca excess (%) than G ×N (16.87 % for RP-20 and 14.23 % for G ×N). Moreover, aeration significantly increased ⁴⁴Ca excess (%) in the shoots compared to unaerated, for both plant materials (17.40 % for aerated and 13.7 % for unaerated). In the whole plantlet, no significant differences were found in rootstock and conditions. Cadff (%) followed the same patterns as ⁴⁴Ca excess (%) due to the fact that in every bioreactor the composition of the culture medium contained the same Ca and ⁴⁴Ca concentrations (Fig. 6).

4. Discussion

Peach rootstocks have been widely selected for their impact on fruit quality and productivity. However, compared to other fruit species such as apples and tomatoes (Ho et al., 1993; del Amor et al., 2006), the nutrient uptake by peach rootstocks has not been as thoroughly characterized. Among the limited number of studies, peach rootstock evaluation has been focused on the fruit and leaf mineral content in productive trees (Reighard et al., 2012; Mestre et al., 2015). In this study, we conducted a comprehensive characterization of the plant Fig. 5. Differential responses of rootstocks G ×N (black circles and continuous lines) and RP-20 (gray squares and dashed lines), under aerated or unaerated conditions (abscissa axis), for ⁴⁴Ca (mg g⁻¹DW) (A, B and C) and ⁴⁴Ca (mg) (D, E and F) on roots (A and D), shoots (B and E) and the whole plantlet (C and F). A two- way ANOVA was used to test the main effects of conditions (C) and rootstocks (R). Statistical significance was assumed at p <0.05*.

(7)

material using an in vitro approach within a short time frame. This approach enabled us to assess the root Ca uptake capacity independently of scion interactions in the GreenTray® system. Thus, the methodology presented herein represents a novel approach to clonal in vitro selection based on Ca uptake efficiency and could be extended to other nutrients.

The main factor influencing root Ca uptake was found to be the rootstock clone, rather than the aeration condition during culture in the bioreactor. It is worth mentioning that G ×N exhibited higher levels of

44Ca concentration and content in roots, suggesting a greater capacity for Ca uptake compared to RP-20. Additionally, the results showed that G ×N presented a higher water content in roots compared to RP-20, what could be related to a greater water uptake capacity. These differences could be attributed to their differential vegetative growth. RP-20 is classified as a dwarfing rootstock (Pinochet et al., 2010; Opazo et al., 2020).

On the other hand, G ×N is a rootstock characterized by a higher yield production and trunk cross section in almond cultivars (Lordan et al., 2019), which may be associated with higher nutritional re- quirements. This implies that G ×N rootstock would require a higher Ca fertilization compared to RP-20. Our results are in line with Aras et al.

(2021), who evaluated the tolerance of the peach rootstocks G ×N and GF-677 under Ca deficiency, concluding that G ×N exhibited better tolerance to Ca deficiency.

The ability of G ×N to uptake more Ca could be related to the higher capacity of transpiration in the aerial part under in vitro conditions.

According to our results, there was a correlation between the total plantlet ⁴⁴Ca excess (%) and the FSS (mm²) for both rootstocks under aerated conditions, (G ×N: R²=0.997, p =0.0374; RP-20: R²=1, p =

0.0096). Besides, we observed that G ×N showed higher values of foliar area and FSS under aerated conditions. These results would be in line with those from Aras et al. (2021) who observed that the improved tolerance of G ×N was attributed to an increase in stomatal conductance, which was achieved through physiological responses such as the increment in xylem thickness.

Accordingly, previous works under field conditions have reported that G ×N presented a proper adaptation to drought and a high productivity compared to RP-20 and other eight almond rootstocks (Bell- vert et al., 2021). On the other hand, even though RP-20 has been considered as a dwarfing rootstock, it has been reported that it induced a higher stomata density to grafted almond leaves compared to RP-40 (Opazo et al., 2020). This is consistent with our results where RP-20 leaves presented a higher stomata density than G ×N. However, this higher density implies a smaller stomata size, which together with a smaller leaf area result in a lower FSS (Fig. 3D).

It can be inferred based on the results presented herein that the aboveground part of the plant, particularly the leaves, may play an equally or even more significant role than the roots in the assimilation and transport of Ca. The size and density of stomata determine the leaf transpiration of the plant, meaning that larger stomatal area and higher stomatal density result in increased leaf transpiration (Roth-Nebelsick et al., 2009). Transpiration allows internal water movement within the plant, creating a column of water from the roots to the shoot (Brodribb and Holbrook, 2006). The results presented herein indicated that, for the examined rootstocks, stomatal size along the total surface leaf area could be a more significant influence on transpiration than stomatal density.

According to our findings, the Ca uptake in the roots was not Fig. 6. Differential responses of rootstocks G ×N (black circles and continuous lines) and RP-20 (gray squares and dashed lines), under aerated or unaerated conditions (abscissa axis), for ⁴⁴Ca excess (%) (A, B and C) and Cadff (%) (D, E and F) on roots (A and D), shoots (B and E) and the whole plantlet (C and F). A two- way ANOVA was used to test the main effects of conditions (C) and rootstocks (R). Statistical significance was assumed at p <0.05*.

(8)

Scientia Horticulturae 326 (2024) 112718 generally influenced by aeration conditions. Previous studies in apple

trees reported that the amount of ⁴⁴Ca in roots and the whole plant was not influenced by the reduction of transpiration but did affect the pro- portion of ⁴⁴Ca remaining in roots and the ⁴⁴Ca partitioning to aboveground (Kalcsits et al., 2019). These results are consistent with the findings presented in this study, suggesting that a higher root Ca uptake is not only coupled with the water flow.

Interestingly, only under aerated conditions, there was a strong correlation observed between root FW (mg) (not the root DW) and ⁴⁴Ca excess (%) in shoots for both rootstocks (G ×N:R²=0.998, p =0.0269;

RP-20: R²=1, p =0.0127). This suggests that the water content in the roots is closely associated with the Ca transport withing the plant, particularly when the VPD is sufficiently high, such as in aerated conditions. This association can be explained by the fact that an increment in root water content enhances both the apoplastic pathway and the xylem flux (Steudle, 2000), and therefore the Ca transport through this pathway.

The Ca transport withing the plant could also be stimulated by enhancing of plant growth. According to our results, under aerated conditions, the plantlets showed a tendency to increase their growth rate, particularly in the shoots, along with an increase in foliar area.

Concretely, both plant materials responded equally under aeration conditions. This increase of plant growth rate could be related to an increase of transpiration and, consequently, photosynthesis rate. The forced air renewal (aeration) in the bioreactor could enhance plant transpiration, likely due to the increment in VPD by water vapor decrement. Consequently, the aerated plants could increase the photosynthesis rate and accumulate more photoassimilates, increasing the DW (Jones, 1998).

Simultaneously, the periodic removal of environmental water vapor seems to result in an increment of leaf surface what is correlated with the increment in foliar transpiration (Poorter et al., 2012; Pantin and Blatt, 2018). This positive effect of transpiration on photosynthetic capacity and plant growth is attributed to enhanced nutrient uptake and transport (Cramer et al., 2009). Based on the presented results, it can be inferred that plant growth, influenced by transpiration, is the primary driver for Ca accumulation in shoots under aerated conditions and plant growth appears to be the main factor promoting Ca movement withing the aboveground part of the plantlets. In contrast, under unaerated conditions with a low VPD, transpiration and Ca accumulation are decoupled.

5. Conclusion

In conclusion, the present study showed that the G ×N rootstock exhibited a higher capability for Ca uptake compared to RP-20. The choice of rootstock had a significant impact on Ca uptake, highlighting the importance of rootstock selection for nutrient acquisition. On the other hand, the aeration conditions did not influence Ca uptake in the roots, but it did affect the mobilization of Ca within the aboveground part of the plantlet. The results indicated a strong correlation between root FW and Ca transport within the shoot. These findings emphasize the complex interplay between rootstock characteristics, transpiration, and Ca mobilization. Further research is needed to elucidate the underlying mechanisms and to explore the potential implications for improving nutrient uptake and plant performance in agricultural systems.

Declaration of Competing Interest

The authors declare the following financial interests/personal re- lationships which may be considered as potential competing interests:

Francisca Carrasco-Cuello reports financial support was provided by Spain Ministry of Science and Innovation. Ramon Dolcet-Sanjuan has patent #ES2763637B1 issued to IRTA, Spain.

Data availability

Data will be made available on request.

Acknowledgements

Research sponsored by ⁴⁴CaPeach (PID2019-111583RR-I00) Project (“Ministerio de Ciencia e Innovaci´on”) and IRTA. Authors would like to also thank European Social Found (ESF) “ESF invest in your future” for Ph.D. grant PRE2020-095467 (Carrasco-Cuello, F) and Sandra Fran- quesa and Maria Casanovas for the technical support.

References

Aras, S., Keles, H., Bozkurt, E., 2021. Physiological and histological responses of peach plants grafted onto different rootstocks under calcium deficiency conditions. Sci.

Hortic. https://doi.org/10.1016/j.scienta.2021.109967.

Ayars, J.E., Johnson, R.S., Phene, C.J., Trout, T.J., Clark, D.A., Mead, R.M., 2003. Water use by drip-irrigated late-season peaches. Irrig. Sci. https://doi.org/10.1007/

s00271-003-0084-4.

Bellvert, J., Nieto, H., Pelech´a, A., Jofre-Cekaloviˇ ´c, C., Zazurca, L., Miarnau, X., 2021.

Remote sensing energy balance model for the assessment of crop evapotranspiration and water status in an almond rootstock collection. Front. Plant Sci. https://doi.org/

10.3389/fpls.2021.608967.

Bonomelli, C., Fern´andez, V., Capurro, F., Palma, C., Videla, X., Rojas-Silva, X., M´artiz, J., 2022. Absorption and distribution of calcium (45Ca) applied to the surface of orange (Citrus sinensis) fruits at different developmental stages.

Agronomy. https://doi.org/10.3390/agronomy12010150.

Brodribb, T.J., Holbrook, N.M., 2006. Declining hydraulic efficiency as transpiring leaves desiccate: two types of response. Plant Cell Environ. https://doi.org/10.1111/

j.1365-3040.2006.01594.x.

Cantabella, D., Mendoza, C.R., Teixid´o, N., Vilar´o, F., Torres, R., Dolcet-Sanjuan, R., 2022. GreenTray® TIS bioreactor as an effective in vitro culture system for the micropropagation of Prunus spp. rootstocks and analysis of the plant-PGPMs interactions. Sci. Hortic. https://doi.org/10.1016/j.scienta.2021.110622.

Cramer, M.D., Hawkins, H.J., Verboom, G.A., 2009. The importance of nutritional regulation of plant water flux. Oecologia. https://doi.org/10.1007/s00442-009- 1364-3.

del Amor, F.M., Marcelis, L.F., 2006. Differential effect of transpiration and Ca supply on growth and Ca concentration of tomato plants. Sci. Hortic. https://doi.org/10.1016/

j.scienta.2006.07.032.

Dolcet-Sanjuan, R., Mendoza, C.R., 2018. Reactor System For the in Vitro Culture of Plant material, Kit to Transform a Receptacle in an Adequate Reactor For Such system, and Method For the in Vitro Culture of Plant Material With Such Reactor System. Patent No. ES2763637B1; IRTA, Spain.

Getman-Pickering, Z.L., Campbell, A., Aflitto, N., Grele, A., Davis, J.K., Ugine, T.A., 2020. LeafByte: a mobile application that measures leaf area and herbivory quickly and accurately. Methods Ecol. Evol. https://doi.org/10.1111/2041-210X.13340.

Girona, J., Mata, M., Fereres, E., Goldhamer, D.A., Cohen, M., 2002. Evapotranspiration and soil water dynamics of peach trees under water deficits. Agric. Water Manage.

https://doi.org/10.1016/S0378-3774(01)00149-4.

Hanger, B.C., 1979. The movement of calcium in plants. Commun. Soil Sci. Plant Anal.

https://doi.org/10.1080/00103627909366887.

Heuser, A., Eisenhauer, A., Gussone, N., Bock, B., Hansen, B.T., N¨agler, T.F., 2002.

Measurement of calcium isotopes (δ44Ca) using a multicollector TIMS technique.

Int. J. Mass spectrom. https://doi.org/10.1016/S1387-3806(02)00838-2.

Ho, L.C., Belda, R., Brown, M., Andrews, J., Adams, P., 1993. Uptake and transport of calcium and the possible causes of blossom-end rot in tomato. J. Exp. Bot. https://

doi.org/10.1093/jxb/44.2.509.

Iglesias, I., Gin´e-Bordonaba, J., Garanto, X., Reig, G., 2019. Rootstock affects quality and phytochemical composition of ‘Big Top’nectarine fruits grown under hot climatic conditions. Sci. Hortic. https://doi.org/10.1016/j.scienta.2019.108586.

Iglesias, I., Vilardell, P., Bonany, J., Claveria, E., Dolcet-Sanjuan, R., 2004.

Micropropagation and field evaluation of the pear (Pyrus communis L.) IGE 2002′_{, a} new selection of the cultivar Dr. Jules Guyot. J. Am. Soc. Hortic. Sci. https://doi.org/

10.21273/JASHS.129.3.0389.

Jones, H.G., 1998. Stomatal control of photosynthesis and transpiration. J. Exp. Bot.

387–398.

Kalcsits, L., van Der Heijden, G., Reid, M., Mullin, K., 2017. Calcium absorption during fruit development in ‘Honeycrisp’apple measured using 44Ca as a stable isotope tracer. HortScience. https://doi.org/10.21273/HORTSCI12408-17.

Kalcsits, L., van der Heijden, G., Waliullah, S., Giordani, L., 2019. S-ABA-induced changes in root to shoot partitioning of root-applied 44 Ca in apple (Malus domestica Borkh.). Trees. https://doi.org/10.1007/s00468-018-1789-6.

Lordan, J., Zazurca, L., Maldonado, M., Torguet, L., Alegre, S., Miarnau, X., 2019.

Horticultural performance of ‘Marinada’and ‘Vairo’ almond cultivars grown on a genetically diverse set of rootstocks. Sci. Hortic. https://doi.org/10.1016/j.

scienta.2019.108558.

Melgar, J.C., Dichio, B., Xiloyannis, C., 2023. Fertilization and plant nutrient management. Peach. CABI, pp. 129–137. GB.

(9)

Scientia Horticulturae 326 (2024) 112718 Mestre, L., Reig, G., Betran, J.A., Pinochet, J., Moreno, M.A., 2015. Influence of ´

peach–almond hybrids and plum-based rootstocks on mineral nutrition and yield characteristics of ‘Big Top’nectarine in replant and heavy-calcareous soil conditions.

Sci. Hortic. https://doi.org/10.1016/j.scienta.2015.05.020.

Montanaro, G., Dichio, B., Xiloyannis, C., 2010. Significance of fruit transpiration on calcium nutrition in developing apricot fruit. J. Plant Nutr. Soil Sci. https://doi.org/

10.1002/jpln.200900376.

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. https://doi.org/10.1111/j.1399-3054.1962.

tb08052.x.

Opazo, I., Toro, G., Salvatierra, A., Pastenes, C., Pimentel, P., 2020. Rootstocks modulate the physiology and growth responses to water deficit and long-term recovery in grafted stone fruit trees. Agric. Water Manage. https://doi.org/10.1016/j.

agwat.2019.105897.

Pantin, F., Blatt, M.R., 2018. Stomatal response to humidity: blurring the boundary between active and passive movement. Plant Physiol. https://doi.org/10.1104/

pp.17.01699.

Pinochet, J., 2010. ‘Replantpac’(Rootpac® R), a plum–almond hybrid rootstock for replant situations. HortScience 45 (2), 299–301. https://doi.org/10.21273/

HORTSCI.45.2.299.

Poorter, H., Niklas, K.J., Reich, P.B., Oleksyn, J., Poot, P., Mommer, L., 2012. Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and

environmental control. New Phytol. https://doi.org/10.1111/j.1469- 8137.2011.03952.x.

Reig, G., Mestre, L., Betr´an, J.A., Pinochet, J., Moreno, M.A., 2016. Agronomic and ´ physicochemical fruit properties of ‘Big Top’ nectarine budded on peach and plum based rootstocks in Mediterranean conditions. Sci. Hortic. https://doi.org/10.1016/

j.scienta.2016.06.037.

Reighard, G.L., Bridges, W., Rauh, B., Mayer, N.A., 2012. Prunus rootstocks influence peach leaf and fruit nutrient content. In: VII International Symposium on Mineral Nutrition of Fruit Crops. https://doi.org/10.17660/ActaHortic.2013.984.10.

Roth-Nebelsick, A., Hassiotou, F., Veneklaas, E.J., 2009. Stomatal crypts have small effects on transpiration: a numerical model analysis. Plant Physiol. https://doi.org/

10.1104/pp.109.146969.

Schenk, H.J., Jansen, S., H¨oltt¨a, T., 2021. Positive pressure in xylem and its role in hydraulic function. New Phytol. https://doi.org/10.1111/nph.17085.

Steudle, E., 2000. Water uptake by roots: effects of water deficit. J. Exp. Bot. 51 (350), 1531–1542. https://doi.org/10.1093/jexbot/51.350.1531.

White, P.J., Ding, G., 2023. Long-distance transport in the xylem and phloem.

Marschner’s Mineral Nutrition of Plants. Academic Press, pp. 73–104.

Zhu, J., Yu, Q., Xu, C., Li, J., Qin, G., 2018. Rapid estimation of stomatal density and stomatal area of plant leaves based on object-oriented classification and its ecological trade-off strategy analysis. Forests. https://doi.org/10.3390/f9100616.

Differential response to calcium-labelled (44Ca) uptake and allocation in two peach rootstocks in relation to transpiration under in vitro conditions